|
| Status |
Public on Oct 26, 2016 |
| Title |
PR8 infected cells T12h, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
A/Puerto Rico/8/1934 (H1N1) infected
|
| Organism |
Mus musculus |
| Characteristics |
cell line: LA4
viral infection: A/Puerto Rico/8/1934 (H1N1) infected
hours post infection: 12
|
| Treatment protocol |
Our experimental approach was to infect LA4 cells with the three viruses at times t=0 h and t=12 h and harvest RNA for microarray analysis at t=24 h. Controls were mock-inoculated at both time points. Preliminary experiments were done to establish a multiplicity of infection (MOI) for each virus that resulted in comparable numbers of cells positive for viral antigen at 24 h post-infection (Figure S1). Based on this, LA4 cells were inoculated with 3 TCID50/cell RV, 1 PFU/cell PR8, or 3 PFU/cell MHV. Triplicate wells of LA4 cells in 6-well plates were inoculated with each virus diluted in serum-free medium or were mock-inoculated with serum-free medium for 1 h at 37C. Viral inocula were removed and the cells were rinsed twice with serum-free medium. The cells were incubated in Ham's F12K medium with 2% FBS for 12 or 24 h, at which time RNA was isolated. For the 24 h samples, the media were removed and replaced with fresh media 12 h after inoculation.
|
| Growth protocol |
PR8 (A/Puerto Rico/8/1934 (H1N1)), obtained from BEI Resources (NR-3169), was grown and titrated by plaque assay in MDCK (ATCC: CCL-34) cells. MHV, obtained from ATCC (VR-261), was grown and titrated by plaque assay in 17Cl.1 cells (Sturman and Takemoto, 1972) (provided by Dr. Kathryn Holmes, University of Colorado Denver School of Medicine). RV, obtained from ATCC (VR-1645), was grown and titrated by tissue culture infectious dose 50% (TCID50) assay in HeLa cells (ATCC: CCL-2). LA4 (ATCC: CCL-196), a murine lung epithelial cell line, was cultured in Ham's F12K medium (Mediatech, Manassas, VA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from cell cultures using an RNAeasy Plus kit (QIAGEN)
|
| Label |
Cy3
|
| Label protocol |
Microarray analysis was done on RNA samples submitted to the Genomics Resources Core Laboratory at The Institute for Bioinformatics and Evolutionary Studies at the University of Idaho, Moscow, ID
|
| |
|
| Hybridization protocol |
Samples were hybridized to the Roche Nimblegen 12x135k MM9 Microarray (Design 100718_MM9_EXP).
|
| Scan protocol |
Scan protocol not provided.
|
| Description |
P_12_1
|
| Data processing |
The data were processed with the 'oligo' and 'pdInfoBuilder' packages in R using Robust Multichip Average (rma) preprocessing methodology. This strategy allows background subtraction, quantile normalization and summarization (via median-polish).
|
| |
|
| Submission date |
Oct 25, 2016 |
| Last update date |
Oct 26, 2016 |
| Contact name |
James Theodore Van Leuven |
| E-mail(s) |
jvanleuven@uidaho.edu
|
| Organization name |
University of Idaho
|
| Department |
Center for Modeling Complex Interactions
|
| Street address |
709 S Deakin St
|
| City |
Moscow |
| State/province |
ID |
| ZIP/Postal code |
83844 |
| Country |
USA |
| |
|
| Platform ID |
GPL15887 |
| Series (1) |
| GSE89190 |
Transcriptome response of murine alveolar epithelial cells to infection by three unrelated respiratory viruses commonly studied in mouse models |
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